The Morphology of the Male and Female Reproductive

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Zeitschrift/Journal: Arthropod Systematics and Phylogeny

Jahr/Year: 2017

Band/Volume: 75

Autor(en)/Author(s): Kienbaum Katja, Scholtz Gerhard, Becker Carola

Artikel/Article: The morphology of the male and female reproductive system in two species of spider crabs (Decapoda: Brachyura: Majoidea) and the issue of the velum in majoid reproduction 245-260 75 (2): 245– 260 8.9.2017

© Senckenberg Gesellschaft für Naturforschung, 2017.

The morphology of the male and female reproductive system in two species of spider crabs (Decapoda: Brachyura: Majoidea) and the issue of the velum in majoid reproduction

Katja Kienbaum *, §, 1, Gerhard Scholtz 1, 2 & Carola Becker 1, 2, 3

1 Humboldt-Universität zu Berlin, Institut für Biologie, Vergleichende Zoologie, Philippstr. 13, 10115 Berlin, Germany; Katja Kienbaum * [katja. [email protected]]; Gerhard Scholtz [[email protected]]; Carola Becker [[email protected]] — 2 Cluster of Excellence “Image Knowledge Gestaltung”, Humboldt-Universität zu Berlin, Sophienstr. 22a, 10178 Berlin, Germany — 3 Queen’s University Marine Laboratory; 12–13 The Strand, Portaferry, Co. Down; BT22 1PF, UK — § Former name ‘Katja Jaszkowiak’; * Corresponding author

Accepted 04.iv.2017. Published online at www.senckenberg.de/arthropod-systematics on 30.viii.2017.

Editors in charge: Stefan Richter & Klaus-Dieter Klass

Abstract The reproductive system of spider crabs (Majoidea) has raised considerable interest due to the complexity of female sperm storage or- gans. In several majoid species, the seminal receptacle has been described as being divided into a dorsal storage chamber and a ventral fertilization chamber separated by a muscular velum. The velum is supposed to control the amount of sperm used for fertilization and to play an important role in sperm competition. Here, we present a study on the reproductive systems of the two majoid species, Mithraculus sculptus (Lamarck, 1818) and Stenorhynchus seticornis (Herbst, 1788) using various morphological techniques such as µCT scans and 3D-reconstructions, complemented by paraffin histology. The male gonopods of the herein investigated species are similar in their general morpho­logy and in the presence and distribution of setae. The tubular first gonopod holding the ejaculatory canal is much longer than the short and stout second gonopod, which is supposed to function as a piston in the transport of sperm into the female ducts. The female reproductive system of M. sculptus and S. seticornis conforms to that of other Eubrachyura in possessing paired ovaries, oviducts, seminal receptacles, vaginae, and vulvae. Based on our 3D-reconstructions we demonstrate that there is no division of the seminal receptacle into two chambers separated by a velum. In contrast to this, we observed a spatially restricted invagination of the seminal receptacle. A com- parison of our data with those of previous studies, allows for the conclusion that the invagination of the seminal receptacle may have been misinterpreted and mistaken for a velum by other authors. Thus, the division of the seminal receptacle into two chambers separated by a velum is a character which needs to be re-evaluated.

Key words Seminal receptacle, storage chamber, insemination chamber, gonopods, setae, 3D-reconstruction.

1. Introduction

The Majoidea or spider crabs is a very diverse brachy- al. 1992; Porter et al. 2005). However, data concerning uran group, comprising more than 800 species, with a the phylogenetic position of the Majoidea within the Eu- worldwide distribution in marine waters (De Grave et brachyura is sparse and partially contradictory (Brösing al. 2009). It has been proposed to be a basal branching et al. 2006; Tsang et al. 2014). Whilst their monophyly is lineage within the Eubrachyura based on morphologi- widely accepted (Hultgren & Stachowicz 2008; Mahon cal (Jamieson et al. 1995) and molecular data (Spears et & Neigel 2008; Tsang et al. 2014), relationships among

ISSN 1863-7221 (print) | eISSN 1864-8312 (online) 245 Kienbaum et al.: The reproductive system of Majoidea

the majoid groups remain disputed. The only constant is construction, together with established histological tools, the monophyly of the Oregoniidae and their position as our results reveal that seminal receptacles of both studied sister group to the remaining Majoidea (see Hultgren et species are not divided into two separate chambers and al. 2009 and Guinot et al. 2013 for review). The mor- shed doubt on previous interpretations of such a division phology of brachyuran reproductive systems has been and the presence of a velum (Diesel 1989; Sal Moyano et discussed with respect to phylogenetic (Guinot 1977; al. 2010; González-Pisani et al. 2011). The present study McLay & López Greco 2011; Guinot et al. 2013; Becker demonstrates the value of 3D-reconstructions to under- & Scholtz 2017) and functional analyses (Diesel 1989; stand the spatial organization of reproductive systems Beninger et al. 1991). and the need for further studies in order to re-evaluate the The male copulatory system is a complex arrange- majoid reproductive systems. ment of three paired parts, consisting of the first and sec- ond gonopods and the penes. The tubular first gonopod (G1) forms the ejaculatory canal with a basal and distal opening. During copulation, the second gonopod (G2) 2. Materials & methods and the penis are inserted into the G1 through the basal opening. The penis extrudes the sperm into the ejacula- tory canal of the G1. It is then pushed by the G2 into 2.1. Material the female genital duct (Bauer 1986). Gonopods contain valuable phylogenetic information (Beninger et al. 1991; The specimens of Stenorhynchus seticornis and Mithra- Beninger & Larocque 1998). This is based on their di- culus sculptus were obtained from commercial vendors verse and specific morphology including the patterns of (www.shop-meeresaquaristik.de). Three females of each setae in combination with the conservative nature of go- species were used for histology and the 3D-reconstruc- nopods (Martin & Abele 1986; Vallina et al. 2014). tion of the reproductive organs. All investigated females The female reproductive system of eubrachyuran crabs were mature and two females of M. sculptus were oviger- consists of paired ovaries, oviducts, seminal receptacles, ous. vaginae, and vulvae. The seminal receptacles play a major role in eubrachyuran reproduction due to their ability to store sperm from one or more males that can be used to 2.2. Histology fertilize several consecutive broods (Cheung 1968). The reproductive morphology of the Majoidea has For the histological analyses, specimens were cold-an­ been addressed in several studies (Diesel 1991; Beni- aes­thetised in a freezer at – 18°C for 15 minutes. Whole nger et al. 1993; Lanteigne et al. 1996; Sainte-Marie specimens were preserved either in Bouin’s solution & Sainte-Marie 1998; Rotllant et al. 2007; González- or in “Susa Heidenhain” (MORPHISTO® Evolutions- Pisani et al. 2011; Antunes et al. 2016). In contrast to forschung und Anwendung GmbH, Frankfurt am Main, other eubrachyurans, a division of the seminal receptacle Germany) for 48 – 73 hours. For decalcification, speci- into two distinct chambers has been described in some mens were treated in Ethylenediaminetetraacetic acid majoids (Diesel 1989). According to this view, a mus- (EDTA) for 48 – 72 hours. Specimens were then dehy- cular diaphragm, the velum, separates the seminal re- drated through a series of ethanol solutions and infiltrat- ceptacle into a dorsal secretory “storage chamber” filled ed (Shandon Hypercenter XP, Thermo Fisher Scientific, with sperm and a ventral cuticle “insemination chamber” Waltham, Massachusetts, USA) and embedded with par- with the oviduct junction (Diesel 1989). The putative affin. Sections were prepared at 6 – 8 µm using a rotary existence of a velum has stimulated inferences on the microtome Leica RM2255 (Leica Microsystems GmbH, reproduction of spider crabs in terms of the location of Wetzlar, Germany). All histological sections were stained fertilization (Diesel 1991) and sperm competition of eu- with the trichromatic Masson-Goldner “light green” brachyurans in general (McLay & López Greco 2011). (MORPHISTO®, Frankfurt am Main, Germany). To date, it is not clear which majoid groups actually pos- sess a velum and whether all structures referred to as a velum (Sal Moyano et al. 2010; González-Pisani et al. 2.3. Scanning electron microscopy (SEM) 2011) are really similar to its original description and definition D( iesel 1989). The dissected gonopods of two male specimens of each In this study, we investigate the male and female re- species were cleaned manually after an ultrasonic bath. productive morphology of the majoids Stenorhynchus The first and second gonopods were critical point dried seticornis (Herbst, 1788), belonging to the Inachoididae, (Bal-Tec CPD 030, Balzers, Liechtenstein) and sputter and Mithraculus sculptus (Lamarck, 1818) of the Ma- coated with a gold layer (Bal-Tec SCD 005, Balzers, jidae. Our main goal is to re-evaluate characters of the Liechtenstein). The micrographs were taken using a LEO female reproductive system described by Diesel (1989, (Zeiss) 1430 scanning electron microscope (Carl Zeiss 1990, 1991), in particular the division of the seminal Nano Technology Systems GmbH, Oberkochen, Germa- receptacle into two chambers. By means of the latest ny) and images were processed with the software Corel morphological methods such as µCT-scans and 3D-re- DRAW X6 (Corel, Ottawa).

246 ARTHROPOD SYSTEMATICS & PHYLOGENY — 75 (2) 2017

Fig. 1. Arrangement of different setae types along the gonopods. A: First gono­ pod of (1) Mithraculus sculptus and (2) Stenorhynchus seticornis. B: Second go- nopod of (1) Mithraculus sculptus and (2) Stenorhynchus seticornis. — Abbrevia­ tions: ag = apical girdle; dp = distal po- domere; eco = ejaculatory canal opening; i.G2 = insertion of G2; p = penis; pp = proximal podomere; pt = protuberance.

2.4. Micro-computer-tomography (µCT) system. 3D-reconstruction of the µCT scans was carried out by processing image stacks of virtual sections. These One specimen of each species was fixed in “Susa Hei- sections were then edited the same way as the aligned denhain” (MORPHISTO®, Frankfurt am Main, Germa- his­to­logical sections. All images were processed in Corel ny) for 48 – 73 hours and washed repeatedly in 70% etha- DRAW X6 and Corel PHOTOPAINT X6 (Corel, Ottawa). nol. After dissecting the pleon together with the attached gonopods, samples were dehydrated through a series of ethanol solutions. For contrast improvement, samples were immersed in a 1% iodine-ethanol solution for 24 h 3. Results and subsequently critical point dried (Bal-Tec CPD 030, Balzers, Liechtenstein). The samples were X-ray scanned using a Nanotom (Phoenix | x-ray, GE Sensing and In- 3.1. Male gonopods spection Technologies) high resolution µCT system. 3.1.1. Overall morphology

2.5. Image processing and 3D-recon- The seminal duct passes through the coxa of peraeopod struction 5 and emerges through the sexual opening (gonopore) as a penis. The gonopore lies adjacent to the basal part of The reconstruction of three-dimensional (3D) models the first gonopod (G1). The small penis enters the G1 on was carried out with the Amira software (FEI Visualiza- the opposite side of the opening for the second gonopod tion Sciences Group, Bordeaux). Series of histological (G2) (Fig. 1). In both investigated species the tubular first sections were photographed using a stereo microscope gonopod is longer than the stout second gonopod (Fig. 1). Axioskop 2 equipped with a camera Axio Cam HRc and The G1 (Figs. 2, 3) is tripartite (Fig. 2F). The elongat- processed with the Axio Vision 4.3 software (Carl Zeiss ed distal shaft is characterized by the tubular cuticle that Vision GmbH). The images were turned into grey scale forms the ejaculatory canal, whose suture is visible from and aligned. After the alignment, the 3D-reconstruction the outside. The distal, subterminal opening of the ejacu- was carried out by processing image stacks of these vir- latory canal (eco) is directed medially (Figs. 2A,B, 3A). tual sections. The contours of each reproductive structure The proximal opening for the second gonopod (G2) is (differentiated gray scale values) were marked on the formed between overlapping cuticular folds of the proxi- virtual cross section with a polygon, and the polygons mal side of the shaft (Figs. 2C, 3B). Gonopod tegumental then used to calculate a surface model of the reproductive glands or rosette glands (rg) are situated proximally with-

247 Kienbaum et al.: The reproductive system of Majoidea

Table 1. Location and arrangement of types of setae on first gonopod (G1) and second gonopod (G2) of Stenorhynchus seticornis and Mithraculus sculptus; + = present; – = absent.

Location / arrangement on gonopod setae type S. seticornis M. scultus proximal + middle podomere · along the dorsal edges + + + + · surrounding the basal opening for the G1 and penis pappose (Fig. 2C – D; Fig. 3D) · along the basal two-thirds of the dorsolateral edge + – · one row on basal postero-medial edge + – G1 · along the middle of the dorso- and ventro-lateral area simple – + distal podomere · along the distalmost dorso- and ventro-lateral area simple, very short (Fig. 2B) + + · on the dorso-lateral side of the tip + – bifurcate, short (Fig. 3A) · one row along one-third of the dorso-lateral side + – · around the ejaculatory canal opening denticles (Fig. 3A) + + proximal + middle podomere · grouped along the distal edge + + pappose (Fig. 3C) G2 · grouped along the proximal edge – + distal podomere · around the distal apical girdle denticles (Fig. 2E) + + in the shaft of the G1, arranged around its basal open- differ only in some small details. Thus, the results pre- ing (Fig. 2F1 – 3). Several muscle strands are present in sented in the following apply to both species as long as the gonopods connecting the three podomeres (Fig. 2F). not mentioned separately. Within the G1 three muscles are observed (referred to as m1 – m3). While the m1 and m2 have only one origin 3.2.1. Ovary and oviduct and projection, the m3 has two proximal origins on the ventral and dorsal side of the proximal podomere. The ovaries are paired, elongated organs located dor- In Stenorhynchus seticornis the slightly twisted shaft sally in the cephalothorax with two ovary strands run- is bent laterally and tapers distally (Fig. 1A2). Its distal ning along each body half as anterior and posterior lobes. end is somewhat bulbous with a pointed tip (Fig. 3A). The left and right strands connect ventral to the heart. In Mithraculus sculptus the shaft is elongated, Whereas those of M. sculptus are restricted to the thorax, straight, and slightly bent in dorso-lateral direction on the the posterior ovarian lobes of S. seticornis extend into the first quarter of the basal part. The tip is cone-shaped and pleon and are additionally fused posteriorly. elongated on the ventro-lateral side. It is thereby form- The tissues and cell types that line the ovaries and the ing a pointed tip with a wide opening of the ejaculatory oviduct are continuous (Fig. 4A – F). Each strand wherein canal. A small hook like appendix is present on the dorso- the oocytes develop is highly convoluted and internally medial side below the edge of the tip (Fig. 2A,B). lined by a mono-layered epithelium (Fig. 4A – C,F). The The G2 of both species is tripartite as well. The dis- cells that form the oviduct have a cubic shape with round tal podomere has a compact shaft with a smooth surface basally located nuclei, whereas the cells of the ovary are and only few folds along its ventro-medial side (Fig. more elongated and have oval nuclei. Both structures 3C). Along the distal part of the shaft the cuticle is very are externally coated by a thin layer of connective tissue compact and almost completely fills out the entire lu- (Fig. 4D,E – F). men (Fig. 2F4). The G2 has three muscles (referred to Different stages of oocyte development are present as m*1 – m*3). One of them (m*1) is running within the within the ovaries (Fig. 4B,C). The germinative zones, distalmost segment only (Fig. 2F4). where oogonia proliferate, can be distinguished from the In both species an apical girdle (sensu Beninger et adjacent maturation zones where the oocytes develop. al. 1991) surrounds the tip (see dashed lines in Figs. 2E, However, in females with large mature ovaries, the ma- 3C). The G2 of S. seticornis presents a process at the tip ture oocytes are intermingled with strands of germina- that is pointing dorsally. In M. sculptus the tip of the G2 tive zones. Within the germinative zones, batches of oo- bears a central protuberance (Fig. 2E). gonia (10 – 20 µm) with a low amount of cytoplasm and relatively large nuclei, originate (Fig. 4C,F). Previtello- 3.1.2. Setation genic oocytes with a larger proportion of cytoplasm and an irregular cell shape (20 – 120 µm), are also present The types of setae and their distribution along the surface adjacent to the germinative zone (Fig. 4C,F). The ova- of the gonopods are similar in S. seticornis and M. sculp- ries contain no oocytes in early vitellogenesis (meaning tus (see Table 1 and Fig. 1A,B). the stage of the maturing cells, in which the inclusion of yolk has started only recently) but show mature oocytes at sizes of 140 – 300 µm, completely filled with yolk 3.2. Female reproductive system (Fig. 4A – C,E). In several females, oogonia and previ- tellogenic oocytes are also present within the oviduct The females of Mithraculus sculptus and Stenorhynchus close to the connection to the seminal receptacle (Fig. seticornis have very similar reproductive systems that 4D,F).

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Fig. 2. First and second gonopods (G1, G2) of Mithraculus sculptus, SEM pictures (A–E) and 3D-reconstruction based on µCT (F). A: Tip of G1 with detail of denticles surrounding the ejaculatory canal opening. B: Part of G1 with the hook-like appendix. C: Proximal part of the folded cuticle (see *) forms the opening for the G2. D: Pappose setae along basal two thirds of dorsolateral edge. E: Tip of G2 with the apical girdle (including an enlargement of the denticles) and the protuberance. F: 3D-reconstruction of proximal part of G1 (1–3) and G2 (3, 4). Cuticle is presented semi-transparent to allow visibility of the muscle strands and rosette glands within the gonopods. (1) Course of ejaculatory canal indicated by black dashed lines. The position of the penis and its insertion into the G1 is indicated by a semi-transparent grey area and arrow. (2) All podomeres of G1 can additionally be distinguished through the attachment sites of the muscle strands. Form of middle podomere indicated by dashed line. (3) Position of both gonopods. G2 not inserted into G1. (4) Instead of all other muscle strands both attachment sides of the m*1 are situated within the distal podomere. — Abbreviations: ag = apical girdle; de = denticle; dp = distal podomere; eco = ejaculatory canal opening; ha = hook-like appendix; i.G2 = insertion of G2; m1 = ventral muscle bundle within G1 run- ning within the distal podomeres; m2, m3 = two dorsal muscle bundles within G1 that run from the distal to the proximal podomere; m2’, m3’ = two ventral muscle bundles within G1 that run from the distal to the proximal podomere; m*1 = muscle bundle running within distal podomere of G2; m*2, m*3 = muscle bundles within G2 running within the distal podomeres and the middle podomere to the proximal podomere, respectively; mp = middle podomere; p = penis; pa.se = pappose setae; pp = proximal podomere; pt = protuberance; rg = rosette glands; s.se = simple setae; sut = suture; vs.se = very short setae; * = cuticle folding that forms part of insertion site of G2.

249 Kienbaum et al.: The reproductive system of Majoidea

Fig. 3. First and second gonopods (G1, G2) of Stenorhynchus seticornis, SEM pictures. A: Tip of G1 with ejaculatory canal opening (see *) surrounded by denticles (lower enlargement) and bifurcate setae (upper enlargement), suture of folded cuticle clearly visible. B: Proximal part of folded cuticle (see *) that forms the opening for G2. Dashed lines mark middle and proximal podomeres. C: Overview of G2, api- cal girdle indicated by dashed line; * indicates area of longitudinal folds along distal podomere. D: Pappose setae along edge of G1 distal podomere. E: Process at apex of G2. — Abbreviations: ag = apical girdle; am = appendix masculine; bf.se = bifurcate setae; de = denticle; dp = distal podomere; i.G2 = insertion of G2; sut = suture; pa.se = pappose setae; pp = proximal podomere.

3.2.2. Seminal receptacle and parts of the dorsal area is formed by a columnar epi- thelium. The seminal receptacle (SR) is externally coated by con- In M. sculptus, the ventral cuticle-lined part of the nective tissue. Internally, a dorsal secretory area and a SR forms several prominent bulges which protrude into ven­tral cuticle area can be discriminated (Figs. 5A1 – 4,B, the lumen and partly divide it into different areas (Fig. 6A1,­2,B – C). The tissue of the dorsal area is stratified. Due 5A3,4,B). The cuticle occurs also in the dorsal area, to an irregular surface, the cells that form the outer cell where it covers the secretory tissue towards the lumen layer, appear loosely arranged. They are associated with (Fig. 5E). the surrounding connective tissue. The adjoining prolifera­ In some sections the SR of S. seticornis shows a cu- tive cells form the middle section and an increasing de­ ticle structure that transforms into a cuticle bulge that ge­ne­ration of cells towards the lumen of the SR results in protrudes towards the lumen of the ventral area (Fig. the release of secretions (Fig. 5D). The lumen is filled with 6D,F). sperm masses without any apparent layering or divisions of the sperm masses by sperm gel (Figs. 5E,G, 6D). 3.2.3. The vagina The oviduct runs into a thickened portion of the se- cretory tissue (Fig. 6C,G), close to the transition into the The cuticle of the SR is continuous with the cuticle of the cuticle epithelium (Fig. 4D) and to the adjoining vagina vagina. In cross sections the vagina is crescent shaped, (Fig. 5A3). Cuticle folds are present at the transition be- resembling the “concave type” vagina (sensu Hartnoll tween the secretory tissue and the cuticle area of the SR 1968) with the inner wall invaginated into the outer wall (Figs. 5F, 6C,E). The cuticle that lines the SR ventrally occluding the vagina lumen. Two different cuticle layers

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Fig. 4. Ovary and oviduct of Stenorhynchus seticornis (A,B,E) and Mithraculus sculptus (C,D,F). A: 3D-reconstruction of oviduct and parts of ovary based on histological sections showing mature oocytes within oviduct. Black dashed arrows = continuation of tissue; dark dashed line and arrow = orifice to seminal receptacle. B: Histological section through oviduct within ovary. Oogonia enclosed within ovi- duct, the mature oocytes arranged adjacent to it. C: Cellular organisation within ovary and oviduct. Germinative zones arranged in strands in between the mature oocytes. Some haemal vessels can be found. Notice the central nucleus in the mature oocytes. D: Histological lon- gitudinal section of oviduct connecting ovary to seminal receptacle. E: Magnification of two adjacent mature oocytes within ovary. Notice the “follicle cells” that lie between the oocyte cell membrane and a very fine membrane that is present adjacent to the oocytes.F : From right to left: Oogonia and previtellogenic oocytes within oviduct in very close proximity to orifice connecting oviduct and seminal receptacle. The oviduct lies adjacent to the secretory tissue of the seminal receptacle. Towards the seminal receptacle lumen a cuticle bulge covers the secretory tissue. The lumen of the seminal receptacle is filled with sperm mass. Arrows = connective tissue around both structures. —Ab ­ breviations: ct = connective tissue; cu = cuticle; h = haemal vessel; st.sr = secretory tissue of the seminal receptacle; fc = follicle cell; oc = mature oocyte; n = nucleus; oo = oogonia; od = oviduct; po = previtellogenic oocyte; sp = sperm mass; sr = seminal receptacle.

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Fig. 5. Seminal receptacle and associated structures of Mithraculus sculptus. A: Different perspectives of a 3D-reconstruction of seminal receptacle and parts of oviduct based on histological sections. (1) all parts in shaded outlines; (2) secretory tissue transparent; (3) secretory tissue and oviduct transparent; (4) only cuticular parts of reproductive system visible, view from dorso-medial and cuticle slightly tilted in anterior direction. Cuticle of ventral area shows prominent bulges that protrude into the lumen (see arrowheads part 4) and divides it into different compartments. In some areas these bulges cover the secretory tissue towards the lumen (transparent in part 3). The oviduct connection to the seminal receptacle is medio-ventrally (see * in part 3) in very close proximity to the vagina opening (dashed lines in part 3 + 4). B: Idealized schematic drawing of seminal receptacle. Notice that the oviduct lies adjacent to the secretory tissue. Flexible parts of inner vagina wall indicated by grey area within the cuticle (arrowheads indicate cuticle bulges). C: Histological cross section through

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can be distinguished (see Locke 2001; Fig. 7C). The epi- et al. 1991; Becker et al. 2012). In a system with a short cuticle faces the vagina lumen and stains red in Masson’s G2, the G1 is the actual sperm conduit that interacts with trichrome while the procuticle stains blue. The flexible the vagina while the G2 is supposed to have an accessory parts of the vagina wall are equipped with musculature function by moving the sperm distally within the ejacula- and the cuticle herein appears structurally different from tory canal (Beninger et al. 1991). The narrow ejaculatory the remaining procuticle and stain red in Massons tri- canal allows only minor movements of the G2 within the chrome. In both species investigated, the muscle attach- G1. With respect to the compact cuticle at the distal part ment correlates with the flexible parts of the vagina. of the G2, it seems unlikely that the G2 can be stretched In M. sculptus only the inner vagina wall is flexible significantly but it may however act like a seal, as has and connected to the sternum by muscles running diago- also been suggested by Beninger et al. (1991). The cuti- nally to ventro-lateral (Fig. 7A,B,D,E). In S. seticornis, cle folds on its surface (Fig. 3C) might allow the seal to also the outer vagina wall appears flexible towards the be broken by muscular contractions. SR – indicated by red-staining horizontal bands of the procuticle and a muscle attachment (Fig. 7E). Those 4.1.2. Setation and dentation muscles run diagonally ventro-medial to the sternum. In one specimen a sperm plug was found within the vagina The arrangement of the various setal types along the G1 lumen (Fig. 7F). and G2 of the species investigated is widely consistent with data from other majoid studies (see Table 1). The presence of pappose setae (Figs. 2C,D, 3B – D) at the proximal parts of the gonopods has also been described 4. Discussion for C. opilio (Beninger et al. 1991) and for Libinia spino- sa Guérin, 1832 (Sal Moyano et al. 2011). Furthermore, the denticles located on the gonopod tips are present in 4.1. The male gonopods many other majoid males (Beninger et al. 1991; Diesel 1991; Neumann 1996; Sal Moyano et al. 2011) and have 4.1.1. Overall shape and functions in sperm been regarded as homologous structures (Beninger et al. transfer 1991). The denticles surround the distal opening of the ejaculatory canal of the G1 (Fig. 2A,E) and are supposed The distal segment of the first gonopod (G1) of brachy- to rupture spermatophores during sperm transfer (Beni- uran males is folded longitudinally, forming the ejacula- nger et al. 1991; Neumann 1996; Rorandelli et al. 2008). tory canal with a basal and a distal opening (Rorandelli The observation of intact spermatophores in S. seticornis et al. 2008; Sal Moyano et al. 2011; Vallina et al. 2014). by Antunes et al. (2016) and C. opilio by Sainte-Marie During copulation, the second gonopod (G2) and the pe- et al. (2000) however contradicts this assumption. nis are both inserted into the G1 through a basal open- ing. Together, they form a complex copulatory system to transport the sperm masses into the female seminal 4.2. The female reproductive system receptacles through the vagina during copulation. The relative length of the G1 and G2 is variable among the The morphology of the reproductive system in the inves- Brachyura (see McLay & Becker 2015). A short G2 is tigated species follows the general pattern of heterotreme characteristic for the Majoidea (Beninger et al. 1991; eubrachyurans including other Majoidea. The oviducts Diesel 1991; Neumann 1996; Rorandelli et al. 2008; connect the paired ovaries to the likewise paired semi- Sal Moyano et al. 2011; this investigation) and also nal receptacles, which in turn open to the vulvae through present in other eubrachyuran groups (e.g. Ocypodidae: the vaginae. Furthermore, the ovaries, the oviducts, and Lautenschlager et al. 2010; Pinnotheridae: Becker et al. the seminal receptacles are enclosed by connective tissue 2012). The characters present in the investigated G2 of (Figs. 4D,F,G, 5D,E). Mithraculus sculptus and Stenorhynchus seticornis re- Whereas some studies focused only on some aspects semble those of Chionoecetes opilio (O. Fabricius, 1788) of the majoid reproductive system such as the develop- (Beninger et al. 1991). Even though observations of cop- ment of the ovaries (Hinsch & Cone 1969; Rotllant ulations are rare and data concerning the actual move- et al. 2007), the whole reproductive system has been ment of gonopods are lacking, certain hypotheses about comprehensively described in the species Chionoecetes the transport of sperm have been developed (Beninger opilio (Beninger et al. 1988; Beninger et al. 1993; Lan­

seminal receptacle. A dorsal secretory area can be distinguished from a ventral cuticle area. Prominent folds at the transition between the two parts. Arrow = thin cuticle. D: Stratified tissue of secretory area of the seminal receptacle. E: Cuticle lining of seminal receptacle in dorsal area with stretched columnar epithelium. The cuticle bulges partly cover the secretory tissue that lines the dorsal area. F: Prominent cuticle folds at transition between secretory and cuticle area. G: The sperm mass within the seminal receptacle is not arranged in layers. — Abbreviations: cb = cuticle bulge; ce = columnar epithelium; ct = connective tissue; cu = cuticle; da = dorsal area; dc = disintegrating cells; dor = dorsal; cf = cuticle folds; st = secretory tissue; lu = lumen; m = muscle; med = medial; oc = oocyte; od = oviduct; pc = proliferating cells; post = posterior; sp = sperm mass; stn = sternum; va = ventral area; vag = vagina.

253 Kienbaum et al.: The reproductive system of Majoidea

Fig. 6. Seminal receptacle and associated structures of Stenorhynchus seticornis. A: Different perspectives of a 3D-reconstruction of the seminal receptacle and parts of the oviduct based on histological sections. Oviduct and sternum shown transparent. Additionally three nerves coming from ventral nerve cord run along the seminal receptacle in very close proximity. B: 3D-reconstruction of seminal recep- tacle and parts of oviduct based on histological sections. C: Idealized schematic drawing of the seminal receptacle in longitudinal section. Secretory tissue forms the dorsal area and is followed ventrally by cuticle. Flexible parts of vagina indicated by grey dashed line within the cuticle. Muscles attached to both sides of vagina. The line indicates approximate position of cross section presented in Fig. 6D. D: Cross section through seminal receptacle. The cuticle bulge (see *) transforms into a cuticle structure that can be misinterpreted as a velum (see * in Fig. 6F). At opening of oviduct into seminal receptacle the secretory tissue clusters into a much thicker tissue than anywhere else. E: His- tological section of folds appearing on transition between secretory and cuticle areas. F: Cuticle structure that could be misinterpreted as a velum (see *). G: Transition of oviduct into secretory tissue of seminal receptacle. — Abbreviations: ant = anterior; cf = cuticle folds; cu = cuticle; dor = dorsal; endo.st = endo sternit; st = secretory tissue; lu = lumen; med = medial; m = muscle; n = nerve; op = operculum; od = oviduct; sp = sperm mass; vag = vagina; * = cuticle strap that dents into the lumen (compare Fig. 6F and D).

teigne et al. 1996; Sainte-Marie & Sainte-Marie 1998; 1968), Inachus phalangium (Fabricius, 1775) (Diesel Sainte-Marie et al. 2000; Benhalima & Moriyasu 2001), 1989, 1991), Stenorhynchus seticornis (Antunes et al. Hyas coarctatus Leach, 1816 (Hartnoll 1968; Lan­teigne 2016); Maja brachydactyla Balss, 1922 (Rotllant et al. et al. 1996), Hyas araneus (Linnaeus, 1758) (Hart­noll 2007), Libinia spinosa (Sal Moyano et al. 2010; Gon­

254 ARTHROPOD SYSTEMATICS & PHYLOGENY — 75 (2) 2017

Fig. 7. Histological sections of vagina and associated structures of Mithraculus sculptus (A–D) and Stenorhynchus seticornis (E–F). A: Longitudinal section of vagina with attached muscles. The cuticle epithelium forms prominent folds at the transition to the seminal receptacle. The line indicates approximate position of cross section shown in B. B: Cross section of crescent shaped vagina at transition to seminal receptacle. The flexible inner wall is invaginated into the outer wall.C : Detail of the flexible vagina cuticle. Columnar epithelium lined by cuticle. Two cuticle areas can be distinguished: procuticle and epicuticle (the latter facing the lumen). D: Muscle attachment to the flexible cuticle of inner vagina wall. Arrow = fibrous tissue that connects the muscle to the cuticle.E : Cross section of crescent shaped vagina of S. seticornis. Muscles attached on both sides of vagina walls. Seminal receptacle filled with sperm mass.F : Cross section through vagina, with the sperm plug clearly visible in lumen. — Abbreviations: ce = columnar epithelium; cu = cuticle; epi = epicuticle; pro = procuticle; cf = cuticle folds; fl.cu = flexible part of cuticle; st.sr = secretory tissue of the seminal receptacle; m = muscle; sp.pl = sperm plug; sp = sperm mass; sr = seminal receptacle; st = sternum; lu = lumen (of vagina).

255 Kienbaum et al.: The reproductive system of Majoidea

zález-Pisani et al. 2011; Sal Moyano et al. 2011), Leuro- cyclus tuberculosus (H. Milne Edwards & Lucas, 1842)

(González-Pisani et al. 2011). This broad knowledge ? offers the possibility to identify shared characters of vulva closure vulva inner vagina wall inner vagina inner vagina wall inner vagina wall inner vagina inner vagina wall inner vagina inner vagina wall inner vagina the majoid reproductive system (for a summary see wall inner vagina wall inner vagina wall inner vagina Table 2). ? ? – – – – + + + plug vagina sperm 4.2.1. The shape of the ovaries

The ovarian lobes of Mithraculus sculptus correspond to muscle concave/ + concave/ concave/ + concave/ + concave/ + concave/ + concave/ + concave/ concave/ + concave/ concave/ + concave/ the organization of other Brachyura (McLay & Becker +° concave/ 2015) and are consistent with the H-shape pattern, with / oblique general the ovaries restricted to the cephalothorax (see Krol et ? ? al. 1992). The posterior fusion of the ovaries in S. seti- ? species; investigated in all bold = consistently sph packages packages free mass free mass free cornis (referred to as O-shape herein) is linked with an mass free free mass / sph free extension into the pleon. Interestingly, a similar exten- sperm condition sion of the ovaries has been described for species of three other majoid species (Rotllant et al. 2007; González- sph = spermato - lobes; fused ovary O = posteriorly X shape; ? ?

Pisani et al. 2011). Additionally, an extension of ovar- dorsal dorsal / ventral dorsal dorsal / ventral dorsal / ventral dorsal / ventral dorsal / ventral dorsal ian lobes into the pleon has previously been described / ventral dorsal seminal receptacle sperm distribution for thoracotremes of the groups Grapsoidea (de Souza & Silva 2009), Pinnotheridae (Becker et al. 2011) and = interpretation / disputable; = interpretation ehof – folds Cryptochiridae (V et al. 2016). folds folds folds folds* folds* velum st – cu transition transition structure at structure

Although the macroscopic organization is quite simi- velum / folds italics lar, the cell arrangement of the developing oocytes in the ovaries differs from any known description. So far, the germinative zones of heterotreme ovaries were always + + + + + +

described as situated centrally with oocytes wandering oviduct ? intermediate to the periphery during their maturation progress (Hinsch + / intermediate + / ventral chamber + / ventral & Cone 1969; Johnson 1980; Rotllant et al. 2007). In vagina orifice close to ? ? ? ? – – + + + all females investigated in the present study, this usual cgz arrangement is expanded in a more complex manner with

germinative zones and adjacent previtellogenic oocytes ovary O / pleon O / pleon stretching through areas of mature oocytes (Fig. 4A – C). O / pleon O / pleon dimension morphology/ H / carapace H / carapace H / carapace H / carapace The very small ovaries of a freshly spawned female re- H / carapace semble the usual arrangement to some extent but this seems to be due to the stage of the reproductive cycle. This implies that changes, not only in general size, but also in the histology within the ovaries, depend on the female reproductive cycle. The absence of vitellogenic oocytes within the ovaries may indicate a seasonal repro- duction or a rapid vitellogenesis. this study References Hartnoll 1968 Diesel 1989, 1991 4.2.2. The oviduct origin or where do the et al. 2007 Rotllant González-Pisani et al. 2011 Marie & Sainte-Marie 1998

“follicle cells” fit in? Antunes et al. 2016 this study; Hartnoll 1968; Lanteigne et al. 1996 At first sight, “accessory-” or “follicle cells” seem to be distributed irregularly in between the developing oocytes Beninger et al. 1988, 1993; Lanteigne 1996; Sainte- (Fig. 4E). In some studies they have been interpreted as et al. 2010, 2011; González-Pisani 2011 Sal Moyano the cells that surround the developing oocytes (Hinsch & Cone 1969) and form the chorionic membrane (Johnson 1980; de Souza & Silva 2009). Due to their distribution

and arrangement within the ovary and oviduct it might Species Hyas araneus Hyas Libinia spinosa Hyas coarctatus Hyas

be possible that in fact they are not randomly distributed, Chionoecetes opilio Maja brachydactyla Inachus phalangium Mithraculus sculptus Mithraculus Stenorhynchus seticornis Stenorhynchus but the continuous epithelial cells of the convoluting ovi- tuberculosus Leurocyclus duct and ovary strands (Fig. 4F). Given that germinative zones and premature oocytes are also found within the oviduct in very close proximity Group Oregoniidae Inachoididae Inachidae Majidae Epialtidae phore; st = secretory tissue; cu = cuticle; + = yes; – = no; * = with muscle within the folds; ° = plus an additional muscle; folds; ° = plus an additional the within + = yes; – no; * with muscle cu = cuticle; st = secretory tissue; phore; : cgz = central germinative zone; H = or zone; germinative = central cgz — Abbreviations : Majoidea. of investigated system reproductive female of the comparison A 2. Table to the seminal receptacle, the oviduct can be regarded as = no information available.

256 ARTHROPOD SYSTEMATICS & PHYLOGENY — 75 (2) 2017

part of the ovary that forms the connection to the semi- men of the SR and stretches towards the opposite wall nal receptacle (Fig. 4D,F). The structural similarity of (Fig. 5A4). oviduct and ovary and the view that they should not be Thus, it might be necessary to differentiate between treated as separate structures have been previously dis- a velum in the sense of Diesel (1989) and cuticle invagi- cussed by several authors (Hard 1942; Spalding 1942; nations that incompletely divide the ventral area of the Hartnoll 1968; Becker et al. 2011). SR. The oviduct does not form an open tube where it con- Instead of a velum, in most of the investigated ma- nects to the SR of M. sculptus and S. seticornis. There- joid species some cuticle folds are present at the transi- fore, it seems likely that the tissues of the seminal recep- tion between the dorsal and ventral area of the SR (see tacle and oviduct undergo cyclic changes and only form Table 2 for a summary of the hitherto investigated majoid a tube when the female ovulates. This temporary orifice species). These folds can be structurally different. In C. has been reported for the majoid C. opilio (Sainte-Marie opilio and H. coarctatus musculature inserts into the cu- & Sainte-Marie 1998) and the grapsoid Eriocheir sinen- ticle folds (Beninger et al. 1993; Lanteigne et al. 1996), sis H. Milne Edwards, 1853 (Lee & Yamazaki 1990). whereas in all other species muscles are absent. Antunes et al. (2016) detected musculature within the folds in S. 4.2.3. The seminal receptacle and the issue of seticornis. However, this finding has not been confirmed the velum in our study. The presence of cuticle folds seems to be a widely distributed eubrachyuran character (Becker et al. In both investigated species the dorsal area of the SR is 2011; González-Pisani et al. 2011; de Souza et al. 2013). formed by a secretory tissue whose cells release secre- Nevertheless, with the sperm mass being present in the tions and degenerate towards the lumen (Fig. 5D). Secre- entire lumen of the SR, the cuticle folds seem not to limit tory tissues have been described in numerous eubrachy- its dispersion. Thus, a division in a sperm “storage- and uran species showing different dimensions within the SR insemination chamber” as described by Diesel (1989) is (Johnson 1980; Zara et al. 2014; Ewers-Saucedo et al. unlikely. Due to the lack of a velum or other structures 2015; Hayer et al. 2015; de Souza et al. 2017). Interest- such as a bursa (Vehof et al. 2017), an active participation ingly, the arrangement of the secretory tissue cells at the of the female during copulation regarding the amount of proximity to the oviduct connection of the females of M. sperm used for fertilization and control over specific male sculptus and S. seticornis follows a similar pattern as that sperm seems improbable. described as the “holocrine transfer tissue” in Pinnotheri- antunes et al. (2016) observed spermatophores and dae described by Becker et al. (2011) (see also Antunes free spermatozoa in the ventral region of the SR of S. seti- et al. 2016) (Fig. 6C,G). If this pattern is homologous, cornis. In contrast to this, we observed only free sper- this would undoubtedly serve as a useful character but matozoa in M. sculptus and S. seticornis, which might it needs further investigations into this subject to verify be due to differences in the elapsed time since mating. this. The absence of spermatophores and sperm layering in Diesel (1989, 1991) described a division of the SR the seminal receptacle of both species could also be due into two discrete chambers in females of a number of to this. Similar conclusions have been drawn for hyme- majoid species. The dorsal, secretory “storage cham- nosomatids as sperm masses from multiple copulations ber” and the ventral, cuticular “insemination chamber” slowly mix after some time (van den Brink & McLay were interpreted as a key aspect of majoid reproduc- 2009; Klaus et al. 2014). tion and discussed in terms of sperm competition (Die- Concerning the oviduct orifice of eubrachyurans, sel 1989, 1991). According to this view, the velum Diesel (1991) differentiated between a SR of a dorsal- that separates both chambers could allow the female to type and a ventral-type, with the oviduct orifice being control the amount of sperm stored in the dorsal “stor- located opposite to or adjoining the vagina, respectively. age chamber” and of that released into the ventral “in- This differentiation has been widely accepted and further semination chamber” during ovulation. The concept of hypotheses concerning sperm competition were built the velum has been adopted by some authors for other upon it (Diesel 1991; McLay & López Greco 2011). In majoids and a number of eubrachyuran species (e.g., the herein studied species, the oviduct connection with L. spinosa: Sal Moyano et al. 2010; González-Pisani et the secretory tissue of the seminal receptacle is situated al. 2011; Ucides cordatus (Linnaeus, 1763): Sant’Anna somewhat intermediate. Nevertheless, it is situated close et al. 2007). In the present study however, none of the to the cuticle area and the vagina and therefore of the females possesses a velum, which challenges previous ventral type (Figs. 5, 6). With regard to sperm compe- observations. In some sections of S. seticornis a struc- tition, this would indicate a last male precedence. Yet, ture similar to a velum appears but the 3D-reconstruc- since no layering is obvious, it remains unclear if the tion reveals it to be an invagination of a cuticle-lined stored sperm belongs to more than one male. area of the SR wall (see * in Fig. 6D,F), which does not separate it into two chambers. In M. sculptus a struc- 4.2.4. The vagina ture resembling the velum in L. spinosa (González- Pisani et al. 2011) is present, but is just a prominent The continuity of the cuticle in the SR, the vagina, and cuticle bulge that protrudes into the ventral area-lu- the integument suggests an ectodermal origin of all

257 Kienbaum et al.: The reproductive system of Majoidea these structures. The structures of the vagina have been 6. Acknowledgements elaborated in detail by Hartnoll (1968). In his study on brachyuran female genital ducts he recognized four types of vaginae, namely (1) simple, (2) concave and concave We wish to thank the members of the group Vergleichende Zo- with operculum (3) mobile, and (4) immobile. ologie at the Humboldt-Universität zu Berlin, especially Juliane Vehof for helpful conversations throughout the project and Katrin All hitherto investigated majoid species have vaginae Braun for teaching the use of the Amira software to us. We thank of the concave type and the vulva is enclosed by the de- Jutta Zeller and Kristin Mahlow (Museum für Naturkunde, Berlin), flated inner wall and opens only by contraction ofthe Gabriele Drescher and PD Dr. Thomas Stach (Molekulare Parasi- attached muscle. Hartnoll (1968) refers to this type of tologie) for valuable technical assistance. The helpful comments by closure of the genital ducts as operculum. Colin McLay and an anonymous reviewer are thankfully appreciat- ed. Funding: Katja Kienbaum (former name: K. Jaszkowiak) was funded by a doctoral fellowship of the Heinrich-Böll-Stiftung. The project was funded by the cluster of excellence “Image Knowledge Gestaltung” – an interdisciplinary laboratory, base project “Atten- 5. Conclusions tion and Form” at Humboldt-Universität zu Berlin.

Several characters are shared by the species investigated in the present study (see also Tables 1 and 2): 7. References Male gonopods. (1) The G1 is long, slender, tapers dis- tally and forms a bulbous tip. (2) The opening of the ejaculatory canal is subterminal and (3) surrounded by Antunes M., Zara F.J., López-Greco L.S., Negreiros-Fransozo denticles. (4) The gonopod tegumental glands (= rosette M.L. 2016. Morphological analysis of the female reproductive glands) are present in the proximal part of the G1 where system of Stenorhynchus seticornis (Brachyura: Inachoididae) and comparisons with other Majoidea. – Invertebrate Biology the G2 is inserted. (5) The G2 is short and stout and (6) 135: 75 – 86. has longitudinal folds on its distal surface and (7) an api- Bauer R.T. 1986. Phylogenetic trends in sperm transfer and storage cal girdle is present around its distal tip. (8) The penis complexity in decapod crustaceans. – Journal of Crustacean emerges from the gonopore of the fifth coxa and enters Biology 6: 313 – 325. the G1 opposite the G2. Becker C., Brandis D., Storch V. 2011. Morphology of the female reproductive system of European pea crabs (Crustacea, Deca­ Female reproductive system. Within the SR (1) a poda, Brachyura, Pinnotheridae). – Journal of Morphology (mostly) dorsal secretory area can be distinguished from 272: 12 – 26. (2) a (mostly) ventral area lined by cuticle. (3) Both areas Becker C., Türkay M., Brandis D. 2012. The male copulatory sys- are separated by cuticle folds with or without muscle at- tem of European pea crabs (Crustacea, Brachyura, Pinnotheri- tachment. (4) The vagina is always of the concave pattern dae). – Journal of Morphology 273: 1306 – 1318. Becker C., Scholtz G. 2017. Phylogenetic implications of sperm (sensu Hartnoll 1968) and (5) the vulva is enclosed by storage in Podotremata: Histology and 3D-reconstructions of the inner flexible wall of the deflated vaginal tube. spermathecae and gonopores in female carrier crabs (Deca­ In contrast to earlier descriptions of majoid repro- poda: Brachyura: Homoloidea). – Journal of Morphology 278: ductive systems, the species investigated in the present 89 – 105. study lack a division of the SR into a dorsal sperm “stor- Benhalima K., Moriyasu M. 2001. Prevalence of bacteria in the spermathecae of female snow crab, Chionoecetes opilio (Bra­ age chamber” and a ventral “insemination chamber” chyura: Majidae). – Hydrobiologia 449: 261 – 266. separated by a muscular velum. Instead, we observed Beninger P.G., Elner R.W., Foyle T.P., Odense P.H. 1988. Func- invaginations of the cuticle receptacle wall in histologi- tional anatomy of the male reproductive system and the female cal sections and 3D-reconstructions which represent­ no spermatheca in the snow crab Chionoecetes opilio (O. Fab- anatomical or functional division of the SR. In histologi- ricius) (Decapoda: Majidae) and a hypothesis for fertilization. – Journal of Crustacean Biology 8: 322 – 332. cal sections however, those invaginations resemble the Beninger P.G., Elner R.W., Poussart Y. 1991. Gonopods of the data published by Diesel (1989, 1991) and could by mis- majid crab Chionoecetes opilio (O. Fabricius). – Journal of take be interpreted as a velum. At the present stage, it Crustacean Biology 11: 217 – 228. remains unclear whether a divided seminal receptacle is Beninger P.G., Lanteigne C., Elner R.W. 1993. Reproductive pro- a character which is only present in part of the Majoidea cesses revealed by spermatophore dehiscence experiments and by histology, ultrastructure, and histochemistry of the female or whether histological observations of earlier studies reproductive system in the snow crab Chionoecetes opilio (O. have been misinterpreted. Our findings clearly show the Fabricius). – Journal of Crustacean Biology 13: 1 – 16. benefit of 3D-reconstruction to understand the spatial or- Beninger P.G., Larocque R. 1998. Gonopod tegumental glands: a ganisation of reproductive structures and suggest a re- new accessory sex gland in the Brachyura. – Marine Biology consideration of the velum as a majoid character. 132: 435 – 444. Brösing A., Richter S., Scholtz G. 2006. Phylogenetic analysis of the Brachyura (Crustacea, Decapoda) based on characters of the foregut with establishment of a new taxon. – Journal of Zoological Systematics and Evolutionary Research 45: 20 – 32. Cheung T.S. 1968. Trans-molt retention of sperm in the female stone crab, Menippe mercenaria (Say). – Crustaceana 15: 117 – 120.

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